Fig 2: ACTH improves graft survival in CD28KO mice.(A) Comparison of heart allograft survival in CD28KO recipients of BALB/c hearts that received either ACTH (n = 4 mice/group; MST, 54 days) or no treatment (Control; n = 6 mice/group; MST, 14 days). **P < 0.01 by log-rank test. (B) Representative light micrographs of H&E-stained heart allograft sections from CD28KO recipients following either ACTH treatment or no treatment (Control) at day 7. Scale bars: 750 μm, 500 μm, and 75 μm (from top to bottom). (C) Comparison of cellular infiltration and vascular damage of the heart allografts in CD28KO recipients following either ACTH treatment or no treatment (Control). (D) Representative fluorescence micrographs of CD3+ T cell, CD11b+ cell, and Foxp3+ Treg populations in heart allograft sections following ACTH treatment or no treatment (Control: day 7 after transplant). Scale bars: 50 μm. (E) Bar graphs show comparison of MFIs of CD3, Foxp3, and CD11b staining in heart allograft sections following ACTH treatment or no treatment (Control: day 7 after transplant) (n = 3 mice/group). (F) T cell stimulation assay following ACTH treatment or no treatment. (G) Quantification of IFN-γ, IL-1β, IL-6, TNF-α, and RANTES concentrations following ACTH treatment or no treatment (Control) by ELIspot and Luminex assay. Data presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test.

Fig 3: Schematic of effect of ACTH treatment in T cells.Binding of ACTH to MC2R stimulates expression of IL-2Rα and activation of its intracellular signaling pathway, resulting in Foxp3 expression and differentiation of CD4+ T cells into Tregs (Created with BioRender.com).

Fig 4: R-Tf-D-LP4 treatment of DEN-induced HCC eliminates hepatosteatotic pathology, inflammation, and fibrosis. (A-E) Representative liver sections from DEN-treated, R-Tf-D-LP4–untreated, or R-Tf-D-LP4–treated mice stained with Sirius red (A) or anti–α-SMA (B) or anti-F4/80 antibodies (C) showing macrophage infiltration. IF staining for CD3+ and B220 (D), CD4+, CD8+, or FOXP3 (E). The relative staining intensity (%) is presented in each image. The results are from several images from two mice for each antibody staining. (F) Representative liver sections, H&E stained, demonstrate hepatosteatosis pathology in DEN-induced liver tumors that were absent in R-Tf-D-LP4–treated mice.

Fig 5: Combination of ACTH with low-dose CTLA-4–Ig induced long-term heart allograft survival.(A) Comparison of heart allograft survival between C57BL/6 recipients of BALB/c hearts that were treated with a combination of ACTH and CTLA-4–Ig (n = 5 mice/group; MST, 90 days) or CTLA-4–Ig alone (n = 5 mice/group; MST, 63 days). *P < 0.05 by log-rank test. (B) Representative light micrographs of H&E-stained heart allograft sections at day 28 from C57BL/6 recipients treated with a combination of ACTH and CTLA-4–Ig or CTLA-4–Ig alone. Scale bar: 75 μm. (C) Comparison of cellular infiltration and vascular damage of the heart allografts in CD28KO recipients following treatment with a combination of ACTH and CTLA-4–Ig or CTLA-4–Ig alone (n = 3 mice/group). (D) Representative florescence micrographs of CD3+ T cells, CD11b+ cells, and Foxp3+ Tregs in heart allograft sections of CD28KO recipients following treatment with a combination of ACTH and CTLA-4–Ig or CTLA-4–Ig alone. Scale bar: 100 μm. (E) Representative fluorescence micrographs of fibronectin and collagen I fibers in heart allograft sections of CD28KO recipients following treatment with a combination of ACTH and CTLA-4–Ig or CTLA-4–Ig alone. Scale bar: 100 μm. (F) Light micrographs of Verhoeff’s-stained heart allograft sections of CD28KO recipients following treatment with a combination of ACTH and CTLA-4–Ig or CTLA-4–Ig alone. Scale bar: 75 μm. Data presented as mean ± SEM; *P < 0.05, **P < 0.01 by Student’s t test.